@article{mbs:/content/journal/micro/10.1099/mic.0.27915-0, author = "Das, Gautam and Dineshkumar, T. K. and Thanedar, Swapna and Varshney, Umesh", title = "Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli", journal= "Microbiology", year = "2005", volume = "151", number = "6", pages = "1741-1750", doi = "https://doi.org/10.1099/mic.0.27915-0", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.27915-0", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", keywords = "Cm, chloramphenicol", keywords = "CAT, chloramphenicol acetyl transferase", keywords = "Amp, ampicillin", keywords = "Kan, kanamycin", keywords = "Tc, tetracycline", abstract = " Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA ‘suppressor’. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA1 fMet, and a distantly located metY gene encodes a variant, tRNA2 fMet. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA2 fMet with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA + host background. The mutations were observed when the plasmid-borne gene encoded tRNA2 fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA1 fMet genes in E. coli in safeguarding their intactness, are discussed.", }